Oilseed brassica containing an improved fertility restorer gene for ogura cytoplasmic male sterility

ABSTRACT

The invention is a Brassica plant comprising a homozygous fertility restorer gene for ogura cytoplasmic male sterility, in addition to oilseed, meal and oil produced from the plant, and the use of oilseed of the plant for preparing oil and/or meal. Upon pollination, the plant yields oilseeds having a total glucosinolate content of not more than  30¼  mol/gram, not more than  25¼  mol/gram or not more than  20¼  mol/gram and, optionally, an erucic acid content of no more than two percent by weight based upon the total fatty acid content. The Brassica plant may be Brassica napus. Brassica campestris, or Brassica juncea.

BACKGROUND OF THE INVENTION

[0001] Oilseed from Brassica plants is an increasingly important crop.As a source of vegetable oil, it presently ranks behind only soybeansand palm in commercial importance and it is comparable with sunflowers.The oil is used both as a salad oil and as a cooking oil.

[0002] In its original form, Brassica oil, known as rapeseed oil, washarmful to humans due to its relatively high level of erucic acid.Erucic acid is commonly present in native cultivars in concentrations of30 to 50 percent by weight based upon the total fatty acid content. Thisproblem was overcome when plant scientists identified a germplasm sourceof low erucic acid rapeseed oil (Stefansson, 1983).

[0003] In addition, plant scientists have attempted to improve the fattyacid profile for rapeseed oil (Robbelen, 1984; Ratledge et al., 1984;Robbelen et al., 1975; and Rakow et al., 1973). These references arerepresentative of those attempts.

[0004] Particularly attractive to plant scientists were so-called“double-low” varieties: those low in erucic acid in the oil and low inglucosinolates in the solid meal remaining after oil extraction (i.e.,an erucic acid content of less than 2 percent by weight based upon thetotal fatty acid content, and a glucosinolate content of less than 30μmol/gram of the oil-free meal). These higher quality forms of rape,first developed in Canada, are known as canola.

[0005] More recently, plant scientists have focused their efforts onreducing the glucosinolate content further, to levels of less than 20μmol/gram of oil-free meal, as verified by quantifying trimethylsilyl(TMS) derivatives (Sosulski and Dabrowski, 1984) for spring canola, orless than 20 μmol/gram of whole, ground seed, as determined by highperformance liquid chromatography (HPLC) (International Organization forStandardization, reference number ISO 9167-1:1992(E)) for winter canola.

[0006] Glucosinolates are sulfur-based compounds that remain in thesolid component of the seed-the solid meal-after the seed has beenground and its oil has been extracted. Their structure includes glucosein combination with aliphatic hydrocarbons (3-butenyl glucosinolate,4-pentenyl glucosinolate, 2-hydroxy-3-butenyl glucosinolate, and2-hydroxy-4-pentenyi glucosinolate) or aromatic hydrocarbons(3-indoylmethyl glucosinolate, 1-methoxy-3-indoyl methyl glucosinolate).Aliphatic glucosinolates are also known as alkenyl glucosinolates.Aromatic glucosinolates are also known as indoles.

[0007] High levels of glucosinolates are undesirable because theyproduce toxic by-products when acted upon by the enzyme myrosinase.Myrosinase is a naturally occurring enzyme present in Brassica species.When Brassica seed is crushed, myrosinase is released and catalyzes thebreakdown of glucosinolates to produce glucose, thiocyanates,isothiocyanate and nitrites. When separated from glucose, these otherproducts are toxic to certain mammals. Isothiocyanate, for example,inhibits synthesis of thryroxine by the thyroid and has otheranti-metabolic effects (Paul et al., 1986). Attempts have been made toinactivate the enzyme myrosinase (using steam, for example). Theseattempts have not been entirely successful.

[0008] Rapeseed possesses high levels of glucosinolates (from 100μmol/gram to 200 μmol/gram of oil-free meal), whereas canola possesseslower levels of glucosinolates (less than 30 μmol/gram of oil-freemeal). The levels of glucosinolates in canola are regulated in manycountries. In Europe, for example, winter canola must have aglucosinolate content of less than 25 μmol/gram of seed at 8.5%moisture, as measured by HPLC. In Canada, spring canola must have aglucosinolate content of less than 30 μmol/gram of oil-free meal at 0%moisture, as measured by TMS. Many countries are requiring even lowerlevels of glucosinolates in order to register canola varieties.

[0009] In developing improved new Brassica varieties, breeders useself-incompatible (SI), cytoplasmic male sterile (CMS) and nuclear malesterile (NMS) Brassica plants as the female parent. In using theseplants, breeders are attempting to improve the efficiency of seedproduction and the quality of the F₁ hybrids and to reduce the breedingcosts. When hybridisation is conducted without using Si, CMS or NMSplants, it is more difficult to obtain and isolate the desired traits inthe progeny (F₁ generation) because the parents are capable ofundergoing both cross-pollination and self-pollination. If one of theparents is a SI, CMS or NMS plant that is incapable of producing pollen,only cross pollination will occur. By eliminating the pollen of oneparental variety in a cross, a plant breeder is assured of obtaininghybrid seed of uniform quality, provided that the parents are of uniformquality and the breeder conducts a single cross.

[0010] In one instance, production of F₁ hybrids includes crossing a CMSBrassica female parent, with a pollen producing male Brassica parent. Toreproduce effectively, however, the male parent of the F₁ hybrid musthave a fertility restorer gene (Rf gene). The presence of a Rf genemeans that the F₁ generation will not be completely or partiallysterile, so that either self-pollination or cross pollination may occur.Self pollination of the F₁ generation to produce several subsequentgenerations is important to ensure that a desired trait is heritable andstable and that a new variety has been isolated.

[0011] One Brassica plant which is cytoplasmic male sterile and is usedin breeding is ogura (OGU) cytoplasmic male sterile (R. Pellan-Delourmeet al., 1987). A fertility restorer for ogura cytoplasmic male sterileplants has been transferred from Raphanus sativus (radish) to Brassicaby Institut National de Recherche Agricole (INRA) in Rennes, France(Pelletier et al., 1987). The restorer gene, Rfl originating fromradish, is described in WO 92/05251 and in Delourme et al., (1991).

[0012] However, this restorer is inadequate in that restorer inbreds andhybrids carrying this Rf gene have elevated glucosinolate levels and therestorer is closely related to a decrease in seed set-the number ofovules per silique-(Pellan-Delourme et al., 1988; Delourme et al.,1994). In the case of hybrids, the glucosinolate levels are elevatedeven when the female parent has reduced glucosinolate content. Theselevels, typically more than 30 μmol/gram of oil-free meal, exceed thelevels of glucosinolates allowable for seed registration by mostregulatory authorities in the world. Thus, the restorer can be used forresearch purposes, but not to develop directly canola-quality commercialhybrid varieties. To date, there is no other source of a restorer offertility for ogura cytoplasmic male sterility available.

[0013] INRA outlines the difficulties associated with obtaining restorerlines with low glucosinolate levels for ogura cytoplasmic sterility(Delourme, et al., 1994; Delourme, et al., 1995). INRA indicates thatthese difficulties are due to the linkage between male fertilityrestoration and glucosinolate content in its breeding material. INRAsuggests that more radish genetic information needs to be eliminated inits restorer lines (Delourme, et al., (1995)). Although improvementshave been made to restorers during the past few years, isozyme studiesperformed on the improved restorer lines indicate that radish geneticinformation still remains around the restorer gene (Delourme et al.,1994).

[0014] INRA has attempted to develop a restorer having decreasedglucosinolate levels. It reported a heterozygous restorer with about 15μmol per gram (Delourme et al., 1995). However, (i) this restorer washeterozygous (Rfrf) not homozygous (RfRf) for the restorer gene, (ii)this restorer was a single hybrid plant rather than an inbred line,(iii) there was only a single data point suggesting that this restorerhad a low glucosinolate level rather than multiple data points tosupport a low glucosinolate level, (iv) there was no data to demonstratewhether the low glucosinolate trait was passed on to the progeny of therestorer, and (v) the restorer was selected and evaluated in a singleenvironment-the low glucosinolate trait was not demonstrated to bestable in successive generations in field trials. INRA has notintroduced commercially any homozygous restorer having low glucosinolatelevels. Its restorer (reported in Delourme et al., 1995) cannot be usedto develop restorer inbreds or single cross hybrids products (where therestorer is used as a male inbred) with decreased glucosinolate levelsfor commercial development.

[0015] Canadian patent application 2,143,781 of Yamashita, et al.,published on Sep. 11, 1995, claims a hybrid breeding method for cropplants in the family Brassicaceae in which an F₁ seed is produced bycrossing the female parent of a self-incompatible male sterile line witha male parent. In one embodiment, the male parent possesses a fertilityrestorer gene. The fertility restorer gene (IM-B) is for MS-N1-derivedcytoplasm and was derived from a winter variety (IM line). This was thencrossed with a spring double-low line (62We). Although this restorer isalleged to result in low glucosinolate levels, it is not a restorer forogura cytoplasmic male sterility.

[0016] Other breeders have attempted to introduce Rf genes from radishinto rapeseed plants by means of intergeneric crossing. However, thesecrosses have not been employed practically. Canadian patent application2,108,230 of Sakai, et al, published on Oct. 12, 1993, claims afertility restorer gene of a Raphanus plant which is introduced into aBrassica plant by cell fusion or intergeneric cross. This applicationdoes not disclose (1) a restorer of ogura cytoplasmic male sterilitywhich maintains decreased glucosinolate levels in the oilseed of an F₁generation or (2) the advantageous use of a restorer to develop restorerinbreds and to develop single cross hybrid combinations for commercialproducts (where the restorer is used as a male inbred).

[0017] To attempt to avoid the high glucosinolate content of INRA'srestorer of ogura cytoplasmic male sterility, INRA and Serasem (UNCAC)have developed a Brassica napus variety called SYNERGY®. SYNERGY is across of ogura cytoplasmic male sterile SAMOURAI (bred by INRA) and malefertile FALCON® (bred by NPZ). FALCON does not carry the restorer genefor ogura cytoplasmic male sterility. Therefore, the F₁ hybrid is malesterile. SYNERGY is sold as a “composite hybrid line” (CHL) whichconsists of a blend of roughly 80% male sterile F₁, hybrid (SYNERGY) and20% male fertile (FALCON), which provides pollen for seed-set on themale sterile F₁ plants in the farmer's field.

[0018] There are a number of difficulties, however, in relying upon acomposite hybrid line. The most important are: (1) that Brassica napusis a self-pollinating species, so under poor pollination conditions(such as prolonged cool, wet weather) there may be inadequate pollenmovement from the male fertile plants to the F₁ hybrid plants, resultingin poor seed set and yield, and (2) that the F₁, hybrid plants are morevigorous than the FALCON plants, so the former may outcompete thelatter, resulting in too little pollen being available for optimal seedset and yield on the F₁ plants.

[0019] To date, no one has been able to develop an improved restorerhaving a homozygous (fixed) restorer gene (RfRf) for ogura cytoplasmicmale sterility whose oilseeds have low glucosinolate levels. Therestorer must be homozygous (RfRf) so that it can be used to developrestorer inbreds or, as male inbreds, in making single cross hybridcombinations for commercial product development. Ideally, glucosinolatelevels would be well below those set out in standards for canola invarious countries. That way, breeders could use the improved restorer toproduce Brassica inbreds and hybrids having oilseeds with lowglucosinolate levels. This would benefit farmers, who could then plantBrassica hybrids which, following pollination, would yield oilseedshaving low glucosinolate levels and other desirable characteristics.

[0020] In many countries, oilseeds produced by farmers for crushing orfor export are not checked for their glucosinolate content. Sometimes aparticular lot of canola may have high glucosinolate content, resultingin contamination of the bulk grain to which the poor quality canola isadded. It would be an improvement if the glucosinolate content ofoilseeds was well below the standards set by various countries in orderto avoid contamination of the bulk grain.

[0021] Thus, there remains a need for an improved Brassica plant whichis a homozygous restorer of fertility for ogura cytoplasmic malesterility and which produces an oilseed with low glucosinolate content.To date, Brassica plants which are restorers of fertility for oguracytoplasmic male sterility (i) have been heterozygous, rather thanhomozygous (fixed), for the restorer trait, or (ii) have not producedoilseeds with low glucosinolate content. Indeed, glucosinolate contentof such oilseeds has been higher than 30 μmol/gram of oil-free meal.

[0022] It is an object of the present invention to provide an improvedmature Brassica plant which is a homozygous restorer for oguracytoplasmic male sterility and which has a glucosinolate content of lessthan 30 μmol/gram of seed. This restorer could be used to producerestorer inbreds or hybrids with low glucosinolate content. This wouldallow production of fully-restored, single cross hybrids withgenetically-low glucosinolate content in both the hybrid seed and in theoilseed harvested from the hybrid plants.

[0023] It is an object of the present invention to provide a Brassicaoilseed of the Brassica plant containing a nuclear restorer for oguracytoplasmic male sterility and having an improved glucosinolate level.

[0024] It is another object of the present invention to provide improvedBrassica inbred lines, using the restorer. Another object is to use therestorer as a male inbred in making single cross hybrid combinations todevelop commercial products.

[0025] It is another object of the present invention to provide an oiland edible vegetable meal having an improved glucosinolate levelfollowing simple crushing and extraction.

[0026] These and other objects and advantages of the invention will beapparent to those skilled in the art from a reading of the followingdescription and appended claims.

SUMMARY OF THE INVENTION

[0027] This invention relates to a Brassica plant comprising ahomozygous fertility restorer gene for ogura cytoplasmic male sterility,wherein upon pollination the plant yields oilseeds having a totalglucosinolate content of not more than 30 μmol per gram, 25 μmol pergram or 20 μmol per gram.

[0028] The oilseed of a Brassica plant comprising a homozygous fertilityrestorer gene for ogura cytoplasmic male sterility and having aglucosinolate content of less than than 30 μmol per gram, 25 μmol pergram or 20 μ)mol per gram, may be used for preparing oil and/or meal.

[0029] This invention also relates to a Brassica plant comprising ahomozygous fertility restorer gene for ogura cytoplasmic male sterility,wherein upon pollination the plant yields oilseeds having (i) a totalglucosinolate content of not more than 30 μmol per gram and an erucicacid content of no more than 2 percent by weight based upon the totalfatty acid content, (ii) a total glucosinolate content of not more than25 μmol per gram and an erucic acid content of no more than 2 percent byweight based upon the total fatty acid content or (iii) a totalglucosinolate content of not more than 20 μmol per gram and an erucicacid content of no more than 2 percent by weight based upon the totalfatty acid content.

[0030] The Brassica plant may be Brassica napus , Brassica campestris orBrassica juncea . It may be designated as 95SN-9369, 96FNW-1792,96FNW-1822, 96FNW-1348, 96FNW-1628 or their sub-lines. The sub-lines maybe selected from a group consisting of 97SN-1650 (sub-line of95SN-9369), 97SN-1651 (sub-line of 95SN-9369), 96FNW1792-03 (sub-line of96FNW-1792), 96FNW1822-07 (sub-line of 96FNW1822) and 96FNW1822-08(sub-line of 96FNW1822).

[0031] An inbred Brassica plant may be produced using this plant. Ahybrid Brassica plant may be produced using this plant. Uponpollination, the inbred or hybrid plant yields oilseed having a totalglucosinolate content of (i) not more than 30 μmol per gram, (ii) notmore than 25 μmol per gram, or (iii) not more than 20 μmol per gram.

[0032] This invention also includes an oilseed of the Brassica plant orfrom the inbred or hybrid Brassica plant. The oilseed may be present asa component of a substantially homogeneous assemblage of oilseeds whichpossess the specified glucosinolate content. Oil of the oilseed is alsopart of this invention. The oilseed may be formed on Brassica napus ,Brassica campestris or Brassica juncea. The mature Brassica oilseed iscapable of yielding an endogenous vegetable oil having a glucosinolatecontent of no more than (i) 30 μmol per gram, (ii) 25 μmol per gram, or(iii) 20 μmol per gram.

[0033] Meal which is substantially oil free and which is produced fromthis oilseed is also part of this invention. The meal has aglucosinolate content of no more than (i) 30 μmol per gram, (ii) 25 μmolper gram, or (iii) 20 μmol per gram.

[0034] This invention also relates to a part of the Brassica plant ofthis invention. The plant part may be selected from a group consistingof nucleic acid sequences (RNA, mRNA, DNA, cDNA), tissue, cells, pollen,ovules, roots, leaves, oilseeds, microspores, vegetative parts, whethermature or embryonic.

[0035] The Brassica plant of this invention may be used to breed a novelBrassica line. The breeding may be selected from a group consisting ofisolation and transformation, conventional breeding, pedigree breeding,crossing, self-pollination, haploidy, single seed descent andbackcrossing.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0036] The invention will now be described in relation to the figures inwhich:

[0037]FIG. 1 illustrates by way of exemplification the formation of newBrassica napus plant material in accordance with the present inventiondesignated 96FNW-1822 as described in greater detail in Example 3.

[0038]FIG. 2 illustrates by way of exemplification the formation of newBrassica napus plant material in accordance with the present inventiondesignated 96FNW-1348 as described in greater detail in Examples 3 and4.

[0039]FIG. 3 illustrates by way of exemplification the formation of newBrassica napus plant material in accordance with the present inventiondesignated 96FNW-1628 as described in greater detail in Example 3.

[0040]FIG. 4 illustrates by way of exemplification the formation of newBrassica napus plant material in accordance with the present inventiondesignated 96FNW-1792 as described in greater detail in Example 1 and 2.

[0041]FIG. 5 illustrates by way of exemplification the formation of newBrassica napus plant material in accordance with the present inventiondesignated 95SN-9369 and its Descendants (97SN-1650, 97SN-1651 andothers) as described in greater detail in Example 6.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0042] Methods for Determining Glucosinolates

[0043] The glucosinolate levels discussed herein are determined inaccordance with two standard procedures, namely (1) high performanceliquid chromatography (HPLC), as described in ISO 9167-1:1992(E), forquantification of total, intact glucosinolates, and (2) gas-liquidchromatography for quantification of trimethylsilyl (TMS) derivatives ofextracted and purified desulfoglucosinolates, as described by Sosulskiand Dabrowski (1984). Both the HPLC and TMS methods for determining theglucosinolate levels discussed herein involve analysis of the solidcomponent of the seed after crushing and oil extraction, (i.e., thede-fatted or oil-free meal).

[0044] Method for Determining Fatty Acid Profile

[0045] The fatty acid concentrations discussed herein are determined inaccordance with a standard procedure wherein the oil is removed from theBrassica oilseeds by crushing and is extracted as fatty acid methylesters following reaction with methanol and sodium methoxide. Next theresulting ester is analyzed for fatty acid content by gas liquidchromatography using a capillary column which allows separation on thebasis of the degree of unsaturation and chain length. This analysisprocedure is described in the work of J.K. Daun et al, 1983, which isherein incorporated by reference.

[0046] Statement of Invention

[0047] A novel edible endogenous vegetable meal is obtained from animproved Brassica oilseed that possesses glucosinolate and, optionally,erucic acid, in a low concentration. The Brassica oilseed contains thehomozygous nuclear restorer gene for ogura cytoplasmic male sterility.Fewer glucosinolates are subjected to the enzyme myrosinase, whichproduces toxic by-products. The novel edible endogenous meal of thepresent invention is formed by the simple crushing of the Brassicaoilseeds and the simple physical separation of the solid component ofthe seed - the solid meal -from the oil component.

[0048] The Brassica oilseeds of the present invention possess aglucosinolate content in the solid component before crushing andextraction of the oil component of less than 30 μmol/gram, and mostpreferably, less than 20 μmol/gram. The glucosinolate content may be anyone or a mixture of alkenyl (3-butenyl glucosinolate, 4-pentenylglucosinolate, 2-hydroxy-3-butenyl glucosinolate, and2-hydroxy-4-pentenyl glucosinolate), MSGL (methylthiobutenylglucosinolate and methylthiopentenyl glucosinolate) and indole(3-indoylmethyl glucosinolate and 1-methoxy-3-indoylmethylglucosinolate). The glucosinolate determination preferably is made onthe air-dry-oil-free solid as measured by the gas liquid chromatography(TMS-based) method of the Canadian Grain Commission. The glucosinolatelevels commonly are made possible by selecting starting materials whichalready are known to form the desired glucosinolate content, and bymaking selections which retain this value following combination with therecited traits.

[0049] Generating Inbred Plants Using Restorer

[0050] The restorer Brassica plant of this invention may be used forinbreeding using known techniques. The homozygous restorer gene of theBrassica plant can be introduced into Brassica inbred lines by repeatedbackcrosses of the Brassica plant. For instance, the resulting oilseedsmay be planted in accordance with conventional Brassica-growingprocedures and following self-pollination Brassica oilseeds are formedthereon. Again, the resulting oilseeds may be planted and followingself-pollination, next generation Brassica oilseeds are formed thereon.The initial development of the line (the first couple of generations ofthe Brassica oilseed) preferably is carried out in a greenhouse in whichthe pollination is carefully controlled and monitored. This way, theglucosinolate content of the Brassica oilseed for subsequent use infield trials can be verified. In subsequent generations, planting of theBrassica oilseed preferably is carried out in field trials. AdditionalBrassica oilseeds which are formed as a result of such self-pollinationin the present or a subsequent generation are harvested and aresubjected to analysis for the desired trait, using techniques known tothose skilled in the art.

[0051] Generating Hybrid Plants Using Restorer as Male Parent

[0052] This invention enables a plant breeder to incorporate thedesirable qualities of an ogura restorer of cytoplasmic male sterilityinto a commercially desirable Brassica hybrid variety. Brassica plantsmay be regenerated from the ogura restorer of this invention using knowntechniques. For instance, the resulting oilseeds may be planted inaccordance with conventional Brassica-growing procedures and followingcross-pollination Brassica oilseeds are formed on the female parent. Theplanting of the Brassica oilseed may be carried out in a greenhouse orin field trials. Additional Brassica oilseeds which are formed as aresult of such cross-pollination in the present generation are harvestedand are subjected to analysis for the desired trait. Brassica napus ,Brassica campestris , and Brassica juncea are Brassica species whichcould be used in this invention using known techniques.

[0053] The hybrid may be a single-cross hybrid, a double-cross hybrid, athree-way cross hybrid, a composite hybrid, a blended hybrid, a fullyrestored hybrid and any other hybrid or synthetic variety that is knowto those skilled in the art, using the restorer of this invention.

[0054] In generating hybrid plants, it is critical that the femaleparent (P₁) that is cross-bred with the ogura restorer (P₂) have aglucosinolate level that is sufficiently low to ensure that the seed ofthe F₁ hybrid has glucosinolate levels within regulatory levels. Theglucosinolate level of the seed harvested from the F₁ hybrid is roughlythe average of the glucosinolate levels of the female parent (P₁) and ofthe male parent (P₂). The glucosinolate level of the hybrid grain (F₂)is reflective of the genotype of the F₁ hybrid. For example, if theobjective is to obtain hybrid grain (F₂) having a glucosinolate level ofless than 20 μmol/gram, and the male parent (ogura restorer) has aglucosinolate level of 15 μmol/gram, the female parent must have aglucosinolate level of less than 25 μmol/gram.

[0055] Generating Plants from Plant Parts

[0056]Brassica plants may be regenerated from the plant parts of therestorer Brassica plant of this invention using known techniques. Forinstance, the resulting oilseeds may be planted in accordance withconventional Brassica-growing procedures and following self-pollinationBrassica oilseeds are formed thereon. Alternatively, doubled haploidplantlets may be extracted to immediately form homozygous plants.

[0057] Vegetable meal

[0058] In accordance with the present invention it is essential that theedible endogenous vegetable meal of the Brassica oilseed containglucosinolate levels of not more than 30 μmol/gram of seed. The femaleparent which can be used in breeding Brassica plants to yield oilseedBrassica germplasm containing the requisite genetic determinant for thisglucosinolate trait is known and is publicly available. For instance,Brassica germplasm for this trait has been available in North Americasince the mid-1970's.

[0059] Representative winter rape varieties that include the geneticmeans for the expression of low glucosinolate content and that arecommercially available in Europe, for example, include, PRESTOL®,EUROL®, BRISTOL® (each available from Semences Cargill), TAPIDOR®,SAMOURAI® (available from Serasem). Representative spring rape varietiesthat include the genetic means for the expression of low glucosinolatecontent and that are commercially available in Canada, for example,include BULLET®, GARRISON® and KRISTANA® (each available from SvalofWeibull).

[0060] Other winter rape varieties that include the genetic means forthe expression of low glucosinolate content and that are commerciallyavailable in Europe include APEX® GOELAND®, FALCON®, LIRAJET®, CAPITOL®and EXPRESS®.

[0061] Also, genetic means for the expression of low glucosinolate traitcan be obtained from American Type Culture Collection, Rockville, Md20852. Seeds were deposited with the ATCC, comprising restorer line97SN-1650 (Accession No. ATCC 97838), 97SN-1651 (Accession No. ATCC97839), 96FNW1792-03 (Accession No. ATCC 209001) and 96FNW1822-07(Accession No. 209002), discussed hereafter. Such low levels ofglucosinolates in the oilseed Brassica serve to impart increasedcommercial value to the meal.

[0062] The edible endogenous vegetable oil of the Brassica oilseedscontains fatty acids and other traits that are controlled by geneticmeans (see US Patent Application entitled, “Improved Oilseed BrassicaBearing An Endogenous Oil Wherein the Levels of Oleic, Alpha-Linolenicand Saturated Fatty Acids Are Simultaneously Provided In An AtypicalHighly Beneficial Distribution Via Genetic Control”, of Pioneer Hi-BredInternational, Inc., W091/15578; and U.S. Pat. No. 5,387,758,incorporated herein by reference.) Preferably erucic acid of theBrassica oilseed is included in a low concentration of no more than 2percent by weight based upon the total fatty acid content that iscontrolled by genetic means in combination with the other recitedcomponents as specified. The genetic means for the expression of sucherucic acid trait can be derived from numerous commercially availablecanola varieties having good agronomic characteristics, such as 46A05,46A65, BOUNTY®, CYCLONE®, DELTA®, EBONY®, GARRISON®, IMPACT®, LEGACY®,LEGEND®, PROFIT®, and QUANTUM®. Each of these varieties is registered inCanada and is commercially available in that country.

[0063] Herbicide Resistance

[0064] As is known to those skilled in the art, it is possible to usethis invention to develop a Brassica plant which is a restorer offertility for ogura cytoplasmic male sterility, produces oilseeds havinglow glucosinolate content and has other desirable traits. Additionaltraits which are commercially desirable are those which would reduce thecost of production of the Brassica crop or which would increase thequality of the Brassica crop. Herbicide resistance, for example, is adesirable trait (see Example 4-1 and 4-2 in which ogura restorer lineswith low glucosinolate content and different types of herbicideresistance have been developed).

[0065] If desired, a genetic means for tolerance to a herbicide whenapplied at a rate which is capable of destroying rape plants which lacksaid genetic means optionally may also be incorporated in the rapeplants of the present invention as described in commonly assigned U.S.Pat. No. 5,387,758, that is herein incorporated by reference.

[0066] Breeding Techniques

[0067] It has been found that the combination of desired 20 traitsdescribed herein, once established, can be transferred into other plantswithin the same Brassica napus , Brassica campestris , or Brassicajuncea species by conventional plant breeding techniques involvingcross-pollination and selection of the progeny. It surprisingly has beendemonstrated that the restorer gene in combination with lowglucosinolate levels is highly heritable, can be transmitted to progeny,and can be recovered in segregating progeny in subsequent generationsfollowing crossing.

[0068] Also, once established the desired traits can be transferredbetween the napus, campestris, and juncea species using the sameconventional plant breeding techniques involving pollen transfer andselection. The transfer of traits between Brassica species, such asnapus and campestris, by standard plant breeding techniques is alreadywell documented in the technical literature. (See, for instance, Tsunadaet al., 1980).

[0069] As an example of the transfer of the desired traits describedherein from napus to campestris, one may select a commercially availablecampestris variety such as REWARD®, GOLDRUSH®, and KLONDIKE®, and carryout an interspecific cross with an appropriate plant derived from anapus breeding line, such as that discussed hereafter (i.e., 95SN-9369).Alternatively, other napus breeding lines may be reliably andindependently developed using known techniques. After the interspecificcross, members of the F₁ generation are self-pollinated to produce F₂oilseed. Selection for the desired traits is then conducted on single F₂plants which are then backcrossed with the campestris parent through thenumber of generations required to obtain a euploid (n=10) campestrisline exhibiting the desired combination of traits.

[0070] In order to avoid inbreeding depression (e.g., loss of vigor andfertility) that may accompany the inbreeding of Brassica campestris ,selected, i.e. BC₁ plants that exhibit similar desired traits whileunder genetic control advantageously can be sib-mated. The resultingoilseed from these crosses can be designated BC₁SIB₁ oilseed.Accordingly, the fixation of the desired alleles can be achieved in amanner analogous to self-pollination while simultaneously minimizing thefixation of other alleles that potentially exhibit a negative influenceon vigor and fertility.

[0071] A representative Brassica juncea variety of low glucosinolatecontent and low erucic acid content into which the desired traits can besimilarly transferred include the breeding lines, 96SJ-2690, 96SJ-2691,and 96SJ-2692.

[0072] Stand of Plants

[0073] The oilseed Brassica plants of the present invention preferablyare provided as a substantially uniform stand of plants that are capableof forming oilseeds providing a meal which exhibits the recited improvedglucosinolate levels. The Brassica oilseeds of the present inventionpreferably are provided as a substantially homogeneous assemblage ofoilseeds which possess the improved glucosinolate levels.

[0074] The improved oilseed Brassica plant of the present invention iscapable of production in the field under conventional oilseed Brassicagrowing conditions that are commonly utilized during oilseed productionon a commercial scale. Such oilseed Brassica exhibits satisfactoryagronomic characteristics and is capable upon self-pollination offorming oilseeds that possess the glucosinolate levels within the mealpresent therein. For the purposes of the present invention,“satisfactory agronomic characteristics” is defined as the ability toyield an oilseed harvest under standard field growing conditions havingglucosinolate levels that are sufficiently low for registration ofcanola varieties (suitable for commercial use).

[0075] The ability to provide in a single edible endogenous vegetablemeal the improved glucosinolate levels of the present invention usingthe ogura restorer of the present invention, is considered to be totallyunexpected. An edible endogenous meal as presently claimed is novel andits production previously eluded all other researchers. One skilled inoilseed Brassica technology reasonably would have concluded that theogura restorer is genetically linked to the gene regulatingglucosinolate levels, i.e. that both genes are on a fragment of RaphanusDNA that has been integrated into a B. napus chromosome. Whereas thereis no alletic variation within the Raphanus DNA fragment, there is noopportunity for a crossover event to separate the Rf gene from the genecoding for elevated glucosinolate content, thus precluding thesimultaneous expression of the restorer and low glucosinolate levels.

[0076] The improved edible endogenous vegetable meal of the presentinvention, in a preferred embodiment, exhibits a satisfactory flavorthat can be described as being generally comparable to that of canolameal. Representative uses of the meal include feed for livestock.Representative uses of the oil include salad, frying, cooking, spraying,and viscous-food product applications. Handling and inventoryconsiderations are greatly simplified since the endogenous vegetablemeal and oil of the present invention fulfills the requirements for awide variety of end uses. Each of these benefits is achieved in astraightforward manner in an endogenous product that inherentlypossesses superior health and nutritional properties.

[0077] The following Examples are presented as specific illustrations ofthe present invention. It should be understood, however, that theinvention is not limited to the specific details set forth in theExamples.

EXAMPLE 1

[0078] Development of the improved OGURA restorer line, 96FNW-1792,including methodology for glucosinolate determination and assessment offixity of the Rf gene (see FIG. 4). Generation: Parent to Fl TimePeriod: November, 1992 to April, 1993 Seed Planted: R40 (originalrestorer source from INRA) and BRISTOL (commercial winter canola fromSemences Cargill, France) Seed Harvested: Fl = 93CWN-867 (= R40 ×BRISTOL) Methods: Parents were grown, and all crossing was carried outin a controlled environment in the greenhouse. R40 (restorer source) wasused as the female parent so that all resulting materials would carrythe OGURA cytoplasm. Generation: F1 to F2 Time Period: May, 1993 toNovember, 1993 Seed Planted: F1 = 93CWN-867 (= R40 × BRISTOL) SeedHarvested: F2 = 94CWN-2133 Methods: F1 plants were grown out toflowering in the greenhouse. Sterile plants were discarded; fertileplants were self-pollinated to produce F2 seed. At maturity, F2 seed washarvested from each F1 plant separately. Each F2 seedlot was screenedfor glucosinolates using the glucose reaction method. The seedlots withthe lowest glucosinolate content were bulked to produce the F2 seed of94CWN-2133 which could be sampled for F3 production. Generation; F2 toF3 Time Period: December, 1993 to June, 1994 Seed Planted: F2 =94CWN-2133 Seed Harvested: F3 = 95FNW-7703 (selected F3 line) Methods:Five hundred F2 plants from the seedlot, 94CWN-2133, were grown out inthe greenhouse. Sterile plants were discarded at flowering, and fertileplants were self-pollinated. At maturity, F3 seed was harvested fromeach F2 plant individually. Each F3 seed line was screened forglucosinolate content, using the Palladium method. Seed of checks, grownin the same greenhouse environment as the F3s, was included in thisanalysis. The F3 seed line, 95FNW-7703, was identified as having lessthan 25 umol/g total glucosinolate content, so was advanced into thefield nursery program. Generation: F3 to F4 Time Period: August, 1994 toJuly, 1995 Seed Planted: F3 = 95FNW-7703 Seed Harvested: F4 = 96FNW1792(selected F4 line) Methods: 95FNW-7703 was planted in the restorerselection nursery in Frouville, France in August, 1994. Followingemergence, there were Ca. 60 plants in a two row nursery plot. Two elitecommercial checks, Bristol and Goeland, were included at frequentintervals in the nursery as checks for comparison.

[0079] At early flowering, 10 single plants within 95FNW-7703 wereself-pollinated by bagging. The fertility of all plants within the linewas assessed by scoring pollen production (male fertility) and seed setwithin developing pods (female fertility). At the end of flowering, thepollination bags were removed.

[0080] At maturity, F4 seed was harvested from each of the 10 selfedplants individually. Seed quality on each of the F4 seedlots wasassessed; lines with shrivelled and/or mouldy seed were discarded.

[0081] Mature, cleaned seed of the remaining F4 lines was analysed forglucosinolate content by the Palladium method. Seed of the Bristol andGoeland checks was harvested, and glucosinolates determined by HPLC.Seed from these checks was included in the Palladium analysis to allowselection of low glucosinolate Rf lines. The average of Bristol andGoeland plus one standard deviation (ca. 18 umol/g total glucosinolates)was used as a culling level. The F4 seed line, 96FNW-1792, had less than18 umol glucosinolate content, and had the lowest glucosinolate contentof any of the 95FNW-7703-derived lines.

[0082] The fertility assessment of 95FNW-7703 identified no sterileplants in a sample of ca. 50 individuals. As the Rf gene is a single,dominant gene, if 95FNW-7703 was segregating for the Rf gene, sterileswould be expected in a frequency of 0.25 with perfect sampling.Statistically, the probability of finding no steriles in a sample of 50if the line is segregating is .000000562. Based on this, we can concludethat 95FNW-7703 is fixed for the Rf gene, meaning that it was derivedfrom an F2 plant which was homozygous Rf.

EXAMPLE 2

[0083] Development of F5 sub-lines of the improved OGURA restorer,96FNW-1792 (continued from Example 1, see FIG. 4). Generation: F4 to F5Time Period: August, 1995 to July, 1996 Seed Planted: F4 = 96FNW-1792Seed Harvested: F5 = 96FNW-1792-02, -03, and -04 Methods: 96FNW-1792 wasplanted in a four row plot in the 1995/96 restorer nursery at Frouville,France. After emergence, there were >100 plants in the nursery plot.BRISTOL and GOELAND were planted as running checks in the nursery.

[0084] During the winter of 95/96, the homozygosity of 20 individualplants within 96FNW-1792 was assessed by determining the PGI-2 isozymephenotype on leaf tissue extract subjected to starch gelelectrophoresis, as described by Delourme and Eber (1992). All plantswere found to be homozygous for the radish PGI-2 phenotype. Since thisphenotype is the product of a PGI-2 allele from radish, which is verytightly linked to the OGURA Rf gene, these results indicate that96FNW-1792, and the 20 specific plants sampled, are fixed for the Rfgene (RfRf).

[0085] At flowering, the 20 selected plants were self-pollinated bybagging. Male and female fertility of all plants within the 96FNW-1792plot were assessed as described in Example 1. No sterile plants werefound in the sample of 100 plants, again indicating that 96FNW-1792 isfixed for the Rf gene. Seed set (number of ovules per silique) waswithin the normal range for Brassica napus . Pollination bags wereremoved at the end of flowering.

[0086] At maturity, seed of each of the 20 plants was harvestedindividually, threshed and cleaned. The lines with the best seed qualitywere selected, and total glucosinolate content on these materials wasdetermined by HPLC. The total gluclosinolate content(indole+MSGL+alkenyl) for three of the selected sub-lines is given inFIG. 4 (F5 generation).

[0087] A sample of 20 plants of each of these three sub-lines was grownout in the greenhouse. Leaf tissue was sampled from each plant withineach sub-line, and PGI-2 isozyme analysis carried out. The resultsindicated that the three lines, and all of the plants within them, arefixed for the Rf gene.

[0088] The three sub-lines (96FNW-1792-02, -03, and -04) are currentlybeing finished as restorer inbreds. They are also being used as maleinbreds in making numerous single cross hybrid combinations forcommercial product development.

EXAMPLE 3

[0089] Development of the improved OGURA restorer lines 96FNW-1822,96FNW-1348, 96FNW-1628 (see FIGS. 1, 2 and 3).

[0090] Generations of plants shown in FIGS. 1, 2 and 3 were grown in thetime periods and using similar source material and methods indicated inExample 1. Glucosinolate and fertility assessments were conducted asindicated in Example 1. Elite commercial checks were included atfrequent intervals as checks for comparison. Again, results of fertilityassessments indicated that a number of sub-lines (as shown in FIGS. 1, 2and 3) were fixed for the Rf gene and had low glucosinolate levels.

[0091] Plants of sub-lines of restorer lines 96FNW-1822 and 96FNW-1348were grown in France during the winter of 1996-97. Sound seed of theseplants was assessed for fertility, and analysed for glucosinolatecontent by HPLC. Fertility observations showed that the sub-lines werefixed for the Rf gene. The HPLC analysis revealed less than 15 uMglucosinolate content in each of these sub-lines. Test crosses wereconducted to assess transmission of the restorer gene. Table 1 belowillustrates the results of the fertility assessments and glucosinolatecontent. TABLE 1 Fertility observations and glucosinolate content ofsub-lines grown in France during the winter of 1996/97. TotalGlucosinolates No. of fertile and sterile plants in a CVN BLN (umol/g byHPLC) sample (France, 1997)* Code Code 1996/97 Inbred Test Cross (New)(Previous) (France) Fertile Sterile Fertile Sterile NW1717 96FNW-1822-28.73 2,000 0 321 1 96FNW-1822-5 10.16 2,000 0 412 1 96FNW-1822-7 8.142,000 0 420 0 96FNW-1822-8 9.82 2,000 0 346 2 NW1712 96FNW-1348-6 14.712,000 0 375 2

EXAMPLE 4

[0092] Development of the improved OGURA restorer line, 96FNW-1348,which combines low glucosinolate content with desirable agronomic traitsand disease tolerance.

[0093] The following table shows performance data for fourfully-restored, single-cross hybrids involving elite female inbreds and96FNW-1348. This data was collected from yield trials at nine Europeanlocations in the 1995/96 testing season. Comparisons are made toSYNERGY, a composite hybrid-line developed by Serasem, France. YieldMaturity Lodging Stem Hybrid: (% Chk) Height (1-9)* (1-9) Disease95-90013 107% 163 5.0 6.5 4.2 95-90002 106% 160 4.0 7.5 5.6 95-90004106% 148 3.8 5.8 4.2 95-90010 105% 165 3.9 7.5 4.6 95-90006 104% 155 4.07.2 4.8 SYNERGY 100% 155 5.4 7.3 4.0

EXAMPLE 5

[0094] Development of improved OGURA restorer lines with lowglucosinolate content, desirable agronomic traits, and herbicideresistance.

5-1: Development of elite OGURA restorer lines with resistance toimidazolinone herbicides:

[0095]1. Produce F₁ of 96FNW1348 (winter low glucosinolate Rfline)×45A71 (spring Pursuit Smart® variety)

[0096]2. Germinate F₁ spray seedlings with 100 g/ha of PURSUIT toconfirm resistance

[0097]3. Produce BC₁F₁ by crossing F₁ to 96FNW1348

[0098]4. Germinate BC₁F₁ spray seedlings with 100 g/ha PURSUIT, select25% of plants with highest level of resistance

[0099]5. Produce BC₁F₂ by selfing selected plants

[0100]6. Germinate BC₁F₂, spray seedlings with 400 g/ha PURSUIT, selfmost resistant plants, harvest F₃S

[0101]7. Germinate F₃ lines, spray with 400 g/ha PURSUIT; select linesin which all plants are resistant, self-pollinate, harvest F4 seed,confirm low glucosinolate content

[0102]8. Continue self-pollination with selection in the nursery;testcross selected imidazolinone resistant (IR) restorer inbreds toelite IR female inbreds, then evaluate low glucosinolate IR hybrids inyield trails

5-2: Development of elite OGURA restorer lines with other forms ofherbicide resistance, i.e. Roundup Ready®), Liberty Link®:

[0103]1. Follow procedures outlined for development of IR inbreds andhybrids, starting with fixed herbicide resistant source

[0104]2. Once elite, low glucosinolate, herbicide resistant restorerlines have been identified, these should be used as parents insubsequent cycles, for crossing with other elite source materials. Newherbicide resistant, low glucosinolate restorer lines can be isolatedfrom these source materials by haploidy, pedigree breeding, orbackcrossing, all of which are methods familiar to those skilled in theart of rapeseed breeding.

EXAMPLE 6

[0105] Development of improved OGURA restorer line, designated 95SN-9369and Descendants (97SN-1650, 97SN-1651 and others) with low glucosinolatecontent and desirable agronomic traits Generation: Parent to F1 (twosteps: C1 = three-way cross, C2 = complex cross) Time Period: C1 =January, 1994 to April, 1994; C2 = May, 1994 to August, 1994 SeedPlanted: C1: female = R40 × TAPIDOR ® (winter); male = BULLET ® (spring)C2: female = C1; male = KRISTINA ® × GARRISON ® Methods: All materialswere grown and crossing was performed in controlled environmentgreenhouses. The R40 × TAPIDOR ® F1 used as the female in C1 was fromthe winter canola breeding program. C2 was made using several fertile C1plants as female, and a bulk pollen sample from several male plants. Thefinal product of C2 was the complex cross F1, ((R40 × TAPIDOR ®) ×BULLET ®) × (KRISTINA ® × GARRISON ®). Generation: F1 to F2 Time Period:September to December, 1994 Seed Planted: F1 = ((R40 × TAPIDOR ®) ×BULLET ®) × (KRISTINA ® × GARRISON ®) Seed Harvested: F2 = 95SN-7805Methods: 32 F1 plants were grown to flowering in the greenhouse andself-pollinated to produce F2 seed. At maturity, F2 seed from each F1plant was harvested separately and analysed for glucosinolate content bythymol method (colorimetric quantification). F2 seedlots with the lowestglucosinolate content, in comparison to a check variety, were selectedfor further breeding. Generation: F2 to F3 Time Period: January, 1995 toApril, 1995 Seed Planted: F2 = 95SN-7805 Seed Harvested: F3 = 95SN-9369(selected F3) Methods: Several hundred F2 plants were grown out in thegreenhouse. At flowering, sterile plants were discarded, and all fertileplants were self-pollinated by bagging. Bags were removed at the end offlowering, and seed was allowed to fully mature on the plants prior toharvest. All F3 seed lines (harvested from individual F2 plants) werescreened for glucosinolate content by the thymol method. The F3 seedline, 95SN-9369 was selected as being among the lowest in glucosinolatecontent. Generation: F3 to F4 Time Period: May, 1995 to August, 1995Seed Planted: F3 = 95SN-9369 Seed Harvested: F4 = 96SN-3077, 96SN-0853,and others (see Figure 5) Methods: A large sample of F3 plants from95SN-9369 was grown to flowering in the greenhouse, and bagged toproduce F4 seed. Bags were removed at the end of flowering; F4 seed washarvested from each F3 plant individually at full maturity. Each F4 seedline (seed harvested from a single F3 plant) was analysed forglucosinolate content by the thymol method. Five F4s were selected forfurther breeding (see Figure 5), including 96SN-3077 and 96SN-0853.Generation: F4 to F5 Time Period: September, 1995 to December, 1995 SeedPlanted: F4 = 96SN-3077, 96SN-0853 and others (see Figure 5) SeedHarvested: F5 = 96SN-3424 (from 96SN-0853), 97SN-0180 (from 96SN-3077)and others (see Figure 5 for details). Methods: Fifteen plants of eachof the selected F4 lines were planted in the greenhouse, along withcheck varieties (for glucosinolate selection). Each plant was bagged atflowering; bags were removed at the end of flowering and seed washarvested from individual plants at full maturity. Each F5 seed line(seed from a single F4 plant) was analysed for glucosinolate content bythe thymol method. The best F5 from each F4 was selected for furtherbreeding. Generation: F5 to F6 (97SN-0180 was not included in thisplanting) Time Period: January, 1996 to April, 1996 Seed Planted: F5 =96SN-3424 and others (see Figure 5) Seed Harvested: F6 = 96SN-9142 (from96SN-3424) and others (see Figure 5 for details) Methods: Fifteen plantsof each of the selected F5 lines were planted in the greenhouse, alongwith check varieties (for glucosinolate selection). Each plant wasbagged at flowering; bags were removed at the end of flowering and seedwas harvested from individual plants at full maturity. Each F6 seed line(seed from a single F5 plant) was analysed for glucosinolate content bythe thymol method. The best F6 from each F5 was selected for furtherbreeding. Generation: Field evaluation - F5 to F6 for 97SN-0180; F6 toF7 for 96SN-9142 Time Period: May, 1996 to August, 1996 Seed Planted: F5= 975N-0180 F6 = 96SN-9142 and others (see Figure 5) Seed Harvested: F6= 97SN-1650 F7 = 975N-1651 (from 965N-9142) and others (see Figure 5 fordetails) Methods: Selected lines were planted in two row plots in anisolation near Hillsburgh, Ontario. After emergence, there were morethan 100 plants per line. At flowering, every plant in a selected linewas scored for fertility/sterility, and 20 plants were bagged to produceselfed seed. Bags were removed at the end of flowering, and seed washarvested at full maturity. Single plants with sound seed were analysedfor glucosinolate content by TMS. Fertility observations showed thatboth 97SN-1651 and 97SN-1650 were fixed (homozygous) for the Rf gene.The TMS analysis revealed less than 15 uM glucosinolate content in bothof these lines (see Figure 5 for precise data). Both lines were observedto have acceptable maturity, standability (lodging resistance) and planttype in the nursery. These lines, and line 97SN-1649, have been advancedinto seed production during the winter of 1996/97 in Chile, where theyare being crossed to several elite ogura male sterile inbreds (females)to produce single cross hybrids. The resulting hybrids were evaluated inmulti-locations trials in western Canada in summer, 1997. Seed from theplants grown in Chile during the winter of 1996/97 were planted inOntario in 1997. Seeds from the resulting plants were harvested in thesummer of 1997 and evaluated for fertility and glucosinolate content.Test crosses were conducted to assess transmission of the restorer gene.The results are shown in Table 2 below.

[0106] TABLE 2 Results of fertility observations and glucosinolateanalysis of lines 97SN- 1649 and 97SN-1650 grown in Chile during 1996-97and Ontario during the spring of 1997. No. of fertile and sterile TotalGlucosino- plants in a sample (On- lates (umol/g by tario, 1997)* CVNBLN TMS) Inbred Test Cross Code Code 96/97 1997 Fert- Ster- Fert- Ster-(New) (Previous) (Chile) (Ontario) ile ile ile ile NS3059 97SN-164911.45 11.32 600 0 38 2 NS3060 97SN-1650 14.40 14.01 600 0 42 0

[0107] A person skilled in the art could use the Brassica plant of thisinvention to develop a Brassica plant which is a restorer of fertilityfor ogura cytoplasmic male sterility, produces oilseeds having lowglucosinolate content and which is resistant to one or more herbicides.Herbicide resistance could include, for example, resistance to theherbicide glyphosate, sold by Monsanto under the trade mark ROUNDUP.Glyphosate is an extremely popular herbicide as it accumulates only ingrowing parts of plants and has little or no soil residue.

[0108] There are two genes involved in glyphosate resistance in canola.One is for an enzyme which detoxifies the herbicide: it is called GOX,glyphosate oxidoreductase. The other is a mutant target gene, for amutant form of EPSP synthase. One skilled in the art could use GOX orCP4 with promoters in canola. Basically, the genes are introduced into aplant cell, such as a plant cell of this invention carrying the restorergene for ogura cytoplasmic male sterility, and then the plant cell growninto a Brassica plant.

[0109] As another example, a person skilled in the art could use theBrassica plant of this invention to develop a Brassica plant which is arestorer of fertility for ogura cytoplasmic male sterility, producesoilseeds having low glucosinolate content and which is resistant to thefamily of imidazoline herbicides, sold by Cyanamid under trade-markssuch as PURSUIT. Resistance to the imidazolines Cyanamid undertrade-marks such as PURSUIT. Resistance to the imidazolines is conferredby the gene AHAS or ALS. One skilled in the art could introduce themutant form of AHAS present in varieties such as the Pioneer® springcanola variety, 45A71, into a Brassica plant which also carries the Rfgene for the ogura cytoplasm. Alternatively, one could introduce amodified form of the AHAS gene with a suitable promoter into a canolaplant cell through any of several methods. Basically, the genes areintroduced into a plant cell, such as a plant cell of this inventioncarrying the restorer gene for ogura cytoplasmic male sterility, andthen the plant cell grown into a Brassica plant.

[0110] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

[0111] The present invention has been described in detail and withparticular reference to the preferred embodiments; however, it will beunderstood by one having ordinary skill in the art that changes can bemade thereto without departing from the spirit and scope thereof.

REFERENCES

[0112] J.K. Daun et al, J. Amer. Oil Chem. Soc., 60:1751-1754 (1983)

[0113] Delourme R., F. Eber, M. Renard. “Breeding Double Low RestorerLines in Radish Cytoplasmic Male Sterility of Rapeseed (Brassica NapusL.).” Proc. 9th Int. Rapeseed Conf. Cambridge. England (1995).

[0114] Delourme R., F. Eber, M. Renard. “Radish Cytoplasmic MaleSterility in Rapeseed: Breeding Restorer Lines with a Good FemaleFertility.” Proc 8th Int. Rapeseed Conf., Saskatoon, Canada. 1506-1510(1991).

[0115] Delourme R., A. Bouchereau, N. Hubert, M. Renard, B.S. Landry.“Identification of RAPD Markers Linked to a Fertility Restorer Gene forthe Ogura Radish Cytoplasmic Male Sterility of Rapeseed ( Brassica napusL.).” Theor. Appi. Gener. 88: 741-748 (1994).

[0116] Delourme, R. and F. Eber. “Linkage Between an Isozyme Marker anda Restorer Gene in Radish Cytoplasmic Male Sterility of Rapeseed(Brassica napus L.).” Theor. Appl. Genet. 85:222-228 (1992).

[0117] International Standard ISO 9167-1:1992(E). “Rapeseed -Determination of glucosinolates content - Part 1: Method usinghigh-performance liquid chromatography.”

[0118] Paul, et al., Theor. Appl. Genet. 72:706-709, (1986).

[0119] Pellan-Delourme, R., Eber, F., Renard, M. 1987. Male fertilityrestoration in Brassica napus with radish cytoplasmic malesterility.Proc. 7th Int. Rapeseed Conf., Poznan, Poland: 199-203.

[0120] Pellan-Delourme, R. and Renard, M. 1988. Cytoplasmic malesterility in rapeseed (Brassica napus L.): Female fertility of restoredrapeseed with “ogura” and cybrids cytoplasms. Genome 30:234-238.

[0121] Pelletier G., C. Primard. “Molecular. Phenotypic and GeneticCharacterization of Mitochondrial Recombinants in Rapeseed.” Proc. 7 thInt. Rapeseed Conf. Poznau. Poland 113-118 (1987).

[0122] Rakow, G. and D.l. McGregor. “Opportunities and Problems inModification of Levels of Rapeseed C₁₈ Unsaturated Fatty Acids.” J. Am.Oil Chem. Soc. 50(10): 400403, (1973).

[0123] Ratledge, Colin, Dawson, Peter and Rattray, James. 1984.Biotechnology for the Oils and Fats Industry. American Oil Chemists'Society, Champaign. 328pp

[0124] Robbelen, Gerhard. “Changes and Limitations of Breeding forImproved Polyenic Fatty Acids Content in Rapeseed.” (Chapter 10) in“Biotechnology for the Oils and Fats Industry” edited by Colin Ratledge,Peter Dawson and James Rattray, American Oil Chemists' Society, (1984).Röbbelen, G. and A. Nitsch. Genetical and Physiological Investigationson Mutants for Polyenic Fatty Acids in Rapeseed, Brassica napus L. Z.Planzenzuchtg., 75: 93-105, (1975).

[0125] Sosulski, F., and K. Dabrowski. “Determination of Glucosinolatesin Canola Meal and Protein Products by Desulfation and CapillaryGas-Liquid Chromatrography.” J. Agric. Food Chem. 32: 1172-1175 (1984).

[0126] Stefansson, B.R. “The Development of Improved RapeseedCultivars.” (Chapter 6) in “High and Low Erucic Acid Rapeseed Oils”edited by John K.G. Kramer, John K.G., Frank D. Sauer. and Wallace J.Pigden. Academic Press Canada, Toronto (1983).

[0127] Tsunada, S, K. Hinata, and Gomex Campo. “Brassica Crops and WildAlleles Biology and Breeding.” Japan Scientific Press, Tokyo (1980).

[0128] The seeds of the subject invention were deposited in the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.,20852, USA Accession Seed No. Deposit Date Brassica napus oleifera97SN-1650  97638 Dec. 23 1996 Brassica napus oleifera 97SN-1651  97839Dec. 23 1996 Brassica napus oteifera 97FNW-1792-03 209001 Apr. 28 1997Brassica napus oleifera 96FNW-1822-07 209002 Apr. 28 1997

We claim:
 1. The use of oilseed of a Brassica plant comprising ahomozygous fertility restorer gene for ogura cytoplasmic male sterilityand having a glucosinolate content of less than 30 μmol per gram, forpreparing oil and/or meal.
 2. The use according to claim 1, wherein theoilseed has a glucosinolate content of less than 25 μmol per gram. 3.The use according to claim 1 or claim 2, wherein the oilseed has aglucosinolate content of less than 20 μmol per gram.
 4. A Brassica plantcomprising a homozygous fertility restorer gene for ogura cytoplasmicmale sterility, wherein upon pollination the plant yields oilseedshaving a total glucosinolate content of not more than 30 μmol per gram5. A Brassica plant comprising a homozygous fertility restorer gene forogura cytoplasmic male sterility, wherein upon pollination the plantyields oilseeds having a total glucosinolate content of not more than 25μmol per gram
 6. A Brassica plant comprising a homozygous fertilityrestorer gene for ogura cytoplasmic male sterility, wherein uponpollination the plant yields oilseeds having a total glucosinolatecontent of not more than 20 μmol per gram
 7. A Brassica plant comprisinga homozygous fertility restorer gene for ogura cytoplasmic malesterility, wherein upon pollination the plant yields oilseeds having atotal glucosinolate content of not more than 30 μmol per gram and anerucic acid content of no more than 2 percent by weight based upon thetotal fatty acid content.
 8. A Brassica plant comprising a homozygousfertility restorer gene for ogura cytoplasmic male sterility, whereinupon pollination the plant yields oilseeds having a total glucosinolatecontent of not more than 25 μmol per gram and an erucic acid content ofno more than 2 percent by weight based upon the total fatty acidcontent.
 9. A Brassica plant comprising a homozygous fertility restorergene for ogura cytoplasmic male sterility, wherein upon pollination theplant yields oilseeds having a total glucosinolate content of not morethan 20 μmol per gram and an erucic acid content of no more than 2percent by weight based upon the total fatty acid content.
 10. TheBrassica plant of claim 1, 2 or 3, and designated by 95SN-9369,96FNW-1792, 96FNW-1822, 96FNW-1348, 96FNW-1628 or their sub-lines. 11.The Brassica plant of claim 7, wherein the sub-lines are selected from agroup consisting of 97SN-1650 (sub-line of 95SN-9369), 97SN-1651(sub-line of 95SN-9369), 96FNW1792-03 (sub-line of 96FNW-1792),96FNW1822-07 (sub-line of 96FNW1822) and 96FNW1822-08 (sub-line of96FNW1822).
 12. The Brassica plant of claim 1, 2, 3, 4, 5, 6, 7 or 8wherein the plant is Brassica napus .
 13. The Brassica plant of claim 1,2, 3, 4, 5, 6, 7 or 8 wherein the plant is Brassica campestris .
 14. TheBrassica plant of claim 1, 2, 3, 4, 5, 6, 7 or 8 wherein the plant isBrassica juncea .
 15. An inbred Brassica plant produced using the plantof claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein upon pollination the plantyields oilseed having a total glucosinolate content of not more than 30μmol per gram.
 16. An inbred Brassica plant produced using the plant ofclaim 1, 2, 3, 4, 5, 6, 7, or 8, wherein upon pollination the plantyields oilseed having a total glucosinolate content of not more than 25μmol per gram.
 17. An inbred Brassica plant produced using the plant ofclaim 1, 2, 3, 4, 5, 6, 7, or 8, wherein upon pollination the plantyields oilseed having a total glucosinolate content of not more than 20μmol per gram.
 18. A hybrid Brassica plant produced using the plant ofclaim 1, 2, 3, 4, 5, 6, 7, or 8, wherein upon pollination the plantyields oilseed having a total glucosinolate content of not more than 30μmol per gram.
 19. A hybrid Brassica plant produced using the plant ofclaim 1, 2, 3, 4, 5, 6, 7, or 8, wherein upon pollination the plantyields oilseed having a total glucosinolate content of not more than 25μmol per gram.
 20. A hybrid Brassica plant produced using the plant ofclaim 1, 2, 3, 4, 5, 6, 7, or 8, wherein upon pollination the plantyields oilseed having a total glucosinolate content of not more than 20μmol per gram.
 21. An oilseed of the plant of claim 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16 or
 17. 22. The oilseed of claim 18which is present as a component of a substantially homogeneousassemblage of oilseeds which possess the specified glucosinolatecontent.
 23. Oil of the oilseed of claim
 18. 24. The oil of claim 20,wherein the oilseed was formed on Brassica napus .
 25. The oil of claim20, wherein the oilseed was formed on Brassica campestris .
 26. The oilof claim 20, wherein the oilseed was formed on Brassica juncea . 27.Meal which is substantially oil free and which is produced using theoilseed of claim
 18. 28. A part of a Brassica plant of claim 1, 2, 3, 4,5, 6, 12, 13, 14, 15, 16 or
 17. 29. The plant part of claim 25, whereinthe part is selected from a group consisting of nucleic acid sequences,tissue, cells, pollen, ovules, roots, leaves, oilseeds, microspores,vegetative parts, whether mature or embryonic.
 30. The plant part ofclaim 26, wherein the nucleic acid sequences are selected from a groupconsisting of RNA, mRNA, DNA, cDNA.
 31. A mature Brassica oilseedcapable of yielding an endogenous vegetable oil having a glucosinolatecontent of no more than 30 μmol per gram.
 32. A mature Brassica oilseedcapable of yielding an endogenous vegetable oil having a glucosinolatecontent of no more than 25 μmol per gram.
 33. A mature Brassica oilseedcapable of yielding an endogenous vegetable oil having a glucosinolatecontent of no more than 20 μmol per gram.
 34. Meal produced from theoilseed of claim 18, having a glucosinolate content of no more than 30μmol per gram.
 35. Meal produced from the oilseed of claim 18, having aglucosinolate content of no more than 25 μmol per gram.
 36. Mealproduced from the oilseed of claim 18, having a glucosinolate content ofno more than 20 μmol per gram.
 37. The Brassica plant of claim 1, 2, 3,4, 5, 6, 12, 13 or 14 for the use of breeding a Brassica line.
 38. Theuse of claim 31, wherein the breeding is selected from a groupconsisting of isolation and transformation, conventional breeding,pedigree breeding, crossing, self-pollination, haploidy, single seeddescent and backcrossing.